Clostridium butyricum Alleviates Enterotoxigenic Escherichia coli K88-Induced Oxidative Damage Through Regulating the p62-Keap1-Nrf2 Signaling Pathway and Remodeling the Cecal Microbial Community.

Clostridium butyricum (CB) can enhance antioxidant capacity and alleviate oxidative damage, but the molecular mechanism by which this occurs remains unclear. This study used enterotoxigenic Escherichia coli (ETEC) K88 as a pathogenic model, and the p62-Keap1-Nrf2 signaling pathway and intestinal microbiota as the starting point to explore the mechanism through which CB alleviates oxidative damage. After pretreatment with CB for 15 d, mice were challenged with ETEC K88 for 24 h. The results suggest that CB pretreatment can dramatically reduce crypt depth (CD) and significantly increase villus height (VH) and VH/CD in the jejunum of ETEC K88-infected mice and relieve morphological lesions of the liver and jejunum. Additionally, compared with ETEC-infected group, pretreatment with 4.4×106 CFU/mL CB can significantly reduce malondialdehyde (MDA) level and dramatically increase superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels in the serum. This pretreatment can also greatly increase the mRNA expression levels of tight junction proteins and genes related to the p62-Keap1-Nrf2 signaling pathway in the liver and jejunum in ETEC K88-infected mice. Meanwhile, 16S rDNA amplicon sequencing revealed that Clostridium disporicum was significantly enriched after ETEC K88 challenge relative to the control group, while Lactobacillus was significantly enriched after 4.4×106 CFU/mL CB treatment. Furthermore, 4.4×106 CFU/mL CB pretreatment increased the short-chain fatty acid (SCFA) contents in the cecum of ETEC K88-infected mice. Moreover, we found that Lachnoclostridium, Roseburia, Lactobacillus, Terrisporobacter, Akkermansia, and Bacteroides are closely related to SCFA contents and oxidative indicators. Taken together, 4.4×106 CFU/mL CB pretreatment can alleviate ETEC K88-induced oxidative damage through activating the p62-Keap1-Nrf2 signaling pathway and remodeling the cecal microbiota community in mice.

Clostridium butyricum Protects IPEC-J2 Cells from ETEC K88-Induced Oxidative Damage by Activating the Nrf2/ARE Signaling Pathway.

Clostridium butyricum (CB) is a naturally occurring probiotic compound that can alleviate the oxidative damage induced by enterotoxigenic Escherichia coli K88 (ETEC K88) in porcine intestinal epithelial (IPEC-J2) cells. In this study, we investigate the molecular mechanism underlying this effect. Based on cell viability, malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPX) assessments, the optimal concentration of ETEC K88 was determined to be 1 × 103 cfu/mL. Viable bacteria counts in cells pretreated with CB and then infected with ETEC K88 show that CB can adhere to IPEC-J2 cells and that optimal adhesion is achieved at the multiple infection index (MOI) of 50 at 3 h of pretreatment. The results of qPCR indicate that although ETEC significantly decreases the expression levels of antioxidant enzymes regulated by NF-E2-related factor 2 (Nrf2) compared to the control group, CB reverses this effect. To confirm that Nrf2 is directly involved in the mechanism by which CB alleviates oxidative stress, siRNA was used to silence the expression of Nrf2 gene in IPEC-J2 cells. Compared to the NC+ETEC and siRNA+ETEC groups, the expressions of SOD1, SOD2, GPX1, and GPX2 in the NC+CB+ETEC and siRNA+CB+ETEC groups are significantly increased at 12 h and 24 h. This shows that CB can reduce ETEC K88-induced oxidative damage in IPEC-J2 cells by activating the expression of antioxidant enzymes implicated in the Kelch-like ECH-associated protein-1- (Keap1-) Nrf2/antioxidant response element (ARE) signaling pathway.

Clostridium butyricum Helps to Alleviate Inflammation in Weaned Piglets Challenged With Enterotoxigenic Escherichia coli K88.

Background: Whether the probiotic Clostridium butyricum (CB) alleviates enterotoxigenic Escherichia coli (ETEC) K88-induced inflammation by regulating the activation of the toll-like receptor (TLR) signaling pathway is not clear, thus, we carried out this study. A total of 72 piglets (average body weight 7.09 ± 0.2 kg) were randomly divided into three groups of 24 piglets per group. Pigs were either fed a daily diet (NC, negative control), a diet tested every day by 1 × 109 CFU/mL ETEC K88 (PC, positive control), or a basal diet supplemented with 5 × 105 CFU/g CB and challenged with ETEC K88 (PC + CB group). Results: Our results showed that CB pretreatment attenuated the effect of ETEC K88 by decreasing C-reactive protein (CRP), which resulted in tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) production. Histological examination revealed that CB pretreatment alleviated intestinal villi injury caused by ETEC K88 challenge. Furthermore, CB pretreatment promoted mRNA expression of the negative regulators of TLR signaling, including myeloid differentiation factor (MyD88), toll-interacting protein (Tollip), and B cell CLL/lymphoma 3 (Bcl-3), in the intestines of ETEC K88-challenged piglets. ETEC K88-induced activation of nuclear factor kappa B (NF-κB) and nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha (IκBα) was attenuated by CB pretreatment. Conclusion: These findings indicate that CB helps to maintain and strengthen the shape of intestinal villi and limits detrimental inflammatory responses, partly by inhibiting toll-like receptor 2 (TLR-2), toll-like receptor 4 (TLR-4), and toll-like receptor 5 (TLR-5) expression and inhibiting NF-κB p65, and promoting IκBα activation and synergism among its negative regulators.

Regulatory Effects of Combined Dietary Supplementation With Essential Oils and Organic Acids on Microbial Communities of Cobb Broilers.

The emergence and spread of antibiotic resistance genes in pathogenic microorganisms have resulted in many countries restricting the use of antibiotics as growth promoters in animal feed. The combined use of essential oils and organic acids can help maintain intestinal health, improve animal growth performance, and alleviate the negative effects of banned antibiotics for certain economically important animals. Although the modes of action for the combined dietary supplementation of essential oils and organic acids such as thymol-citric acid (EOA1) and thymol-butyric acid (EOA2) remain unclear, it is speculated that their activities are achieved through beneficial modulation of gastrointestinal microbial communities and inhibition of pathogen growth. In this study, 16S rDNA amplicon sequencing was used to analyze the effects of treatment with EOA1 and EOA2 on the jejunal, cecal, and fecal microbial communities of Cobb broilers while also evaluating effects over different broiler ages. The intestinal microbial communities of broilers developed with increasing age, and Lactobacillus gradually came to dominate the intestinal communities of treated broilers. Further, the microbial communities of feces were more complex than those of the jejuna and ceca. We systematically elucidate that the longitudinal changes in the intestinal microbial communities of Cobb broiler chickens at different ages. Meanwhile, we found that the addition of EOA1 or EOA2 to the diet: (1) inhibited the proliferation of Ralstonia pickettii and Alcaligenaceae in the jejuna on day 28, (2) promoted the colonization and growth of beneficial bacteria such as Lactobacillus, Clostridia, and Bacteroidia at various growth stages, and (3) enriched the abundance of certain microbiota functions, including biological pathways related to metabolism (e.g., enzyme families). Taken together, the results of this study demonstrate that EOA1 and EOA2 dietary supplementation can affect various microbial metabolic pathways related to the metabolism and absorption of nutrients via regulation of the intestinal microbial community structures of Cobb broilers.

Lactobacillus salivarius alleviates inflammation via NF-κB signaling in ETEC K88-induced IPEC-J2 cells.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) K88 commonly colonize in the small intestine and keep releasing enterotoxins to impair the intestinal barrier function and trigger inflammatory reaction. Although Lactobacillus salivarius (L. salivarius) has been reported to enhance intestinal health, it remains to be seen whether there is a functional role of L. salivarius in intestinal inflammatory response in intestinal porcine epithelial cell line (IPEC-J2) when stimulated with ETEC K88. In the present study, IPEC-J2 cells were first treated with L. salivarius followed by the stimulation of ETEC K88 for distinct time period. ETEC K88 adherent status, pattern recognition receptors (PRRs) mRNA, mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) activation, the release of pro-inflammation cytokines and cell integrity were examined. RESULTS: Aside from an inhibited adhesion of ETEC K88 to IPEC-J2 cells, L. salivarius was capable of remarkably attenuating the expression levels of interleukin (IL)-1ß, tumor necrosis factor-α (TNF-α), IL-8, Toll-like receptor (TLR) 4, nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain-containing protein (NLRP) 3 and NLRP6. This alternation was accompanied by a significantly decreased phosphorylation of p38 MAPK and p65 NF-κB during ETEC K88 infection with L. salivarius pretreatment. Western blot analysis revealed that L. salivarius increased the expression levels of zona occludens 1 (ZO-1) and occludin (P < 0.05) in ETEC K88-infected IPEC-J2 cells. Compared with ETEC K88-infected groups, the addition of L. salivarius as well as extra inhibitors for MAPKs and NF-κB to ETEC K88-infected IPEC-J2 cells had the capability to reduce pro-inflammatory cytokines. CONCLUSIONS: Collectively, our results suggest that L. salivarius might reduce inflammation-related cytokines through attenuating phosphorylation of p38 MAPK and blocking the NF-κB signaling pathways. Besides, L. salivarius displayed a potency in the enhancement of IPEC-J2 cell integrity.

Dietary Clostridium butyricum supplementation modifies significantly the liver transcriptomic profile in weaned piglets.

The addition of probiotics in swine nutrition is known to positively influence both health and growth. The current study investigates differences in the hepatic transcriptome profiles between weaned piglets supplemented with Clostridium butyricum (C. butyricum) and control animals that received no probiotic. The liver is an important metabolic organ that plays a critical role in oxidizing triglycerides for energy production, lipid synthesis and degradation, as well as immune regulation in animals. RNA-Seq analysis was carried out on total RNA harvested from the liver of piglets fed with (n = 3) or without (n = 3) 5 × 105 C. butyricum CFU/g. Compared to the control piglets, 588 of the genes examined (352 up-regulated and 236 down-regulated) were significantly differentially expressed at a fold change > 2 and p < .05 in animals fed with C. butyricum. Quantitative real-time reverse transcription PCR (qRT-PCR) analysis was further used to validate the microarray expression results for 28 genes tested. The functional annotation analyses revealed several genes, processes and pathways with putative involvement in piglet growth and performance. Feeding swine with 5 × 105 C. butyricum CFU/g appears to reinforce their immune status as well as foster the cell cycle and improve the metabolism of carbohydrates, lipids and amino acids. This study provides valuable information about the expression profiles of mRNAs in piglet liver and in-depth functional investigations of these mRNAs that could provide new insights into the molecular networks of growth, immune responses and nutrient metabolism in the porcine liver.

Lactobacillus salivarius, a Potential Probiotic to Improve the Health of LPS-Challenged Piglet Intestine by Alleviating Inflammation as Well as Oxidative Stress in a Dose-Dependent Manner During Weaning Transition.

Intestinal health is a critical issue for piglets during their weaning transition period. Previous reports have emphasized the promise of distinct probiotics in improving the enteric health. Here in this research, a newly isolated Lactobacillus salivarius strain was pretreated to Lipopolysaccharide (LPS)-challenged piglets and its association with integrity of the intestinal barrier coupled with effective dosage were expected to be signified. In the present study, 72 piglets (Landrace × Yorkshiere × Duroc) were randomly allotted to four groups, each group with six replicates. The subjects in the control group were provided with basal diet while those in other tested groups with extra 0.05, 0.1, and 0.2% L. salivarius, respectively. Fourteen days later, LPS was intraperitoneally injected and sodium pentobarbital was then delivered to euthanize those LPS-challenged piglets. An increase of average daily gain and body weight along with an apparent decline of diarrhea rate were observed in L. salivarius-treated groups. Both 0.1 and 0.2% L. salivarius supplement in total diet had the capability to markedly elevate levels of CAT, GSH-Px, SOD, anti-inflammatory cytokine from the serum as well as tight junction proteins (Claudin-1, Occludin, and ZO-1) extracted from intestine in LPS-challenged piglets. These changes were accompanied by the obvious downregulation of D-lactic acid, DAO, MDA and pro-inflammatory mediators in the serum, including IL-1ß, IL-6, IFN-γ, and TNF-α. Meanwhile, the expression levels of TLR2 and TLR4 in spleen and mesenteric lymph nodes were significantly lower whereas the oxidation-related gene, ho-1 was up-regulated with L. salivarius administration. Our findings suggested that relatively high dose L. salivarius (0.1-0.2%) could regulate the progression of inflammatory response and oxidative stress when individuals were exposed to LPS, thus probably offering valuable assistance in restoring barrier function and improving overall performance.

Inhibition of SphK2 Stimulated Hepatic Gluconeogenesis Associated with Dephosphorylation and Deacetylation of STAT3.

BACKGROUND: Sphingosine kinase (SphK) is considered as a potential target for developing novel therapeutics of cancer and other diseases including diabetes. As the major SphK isoform in the liver, much less is known the role of SphK2 involved in regulating hepatic glucose metabolism. METHOD: In this study, RNA interference, real time RT-PCR, western blot and immunoprecipitation method was used to investigate the regulation of SphK2 in hepatic glucose metabolism. RESULTS: Both siRNA and SphK2 inhibitor ABC294640 stimulated expression of gluconeogenetic gene PEPCK and G6Pase but not enzymes of hepatic glycogenolysis, glycolysis and glycogen synthesis. Inhibition of SphK2 also prevented insulin repressed PEPCK and G6Pase expression as well as glucose production levels. Furtherly, inhibition of SphK2 inactivated STAT3 by decreasing both phosphorylation on Tyr705 and acetylation on lysine residue, and led to stimulation of PEPCK and G6Pase expression. Inhibition of SphK2 also prevented IL-6 dependent activation of STAT3 and suppression of PEPCK and G6pase expression both in vitro and in vivo. CONCLUSION: Our study suggests that SphK2 participates in hepatic glucose metabolism related to activation of STAT3.

3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione (K145) ameliorated dexamethasone induced hepatic gluconeogenesis through activation of Akt/FoxO1 pathway.

3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione (K145) is identified as a selective SphK2 inhibitor. It was previously reported as an anti-tumor agent, in this study we demonstrated that K145 was able to regulate hepatic gluconeogenesis and improve glucose intolerance in mice. C57BL/6 mice treated with dexamethasone injection were used as experimental animals, which exhibited impaired glucose tolerance and increased gluconeogenetic enzymes. After K145 treatment, we found that the impairment of glucose tolerance and gluconeogenetic genes mRNA expression were improved. Besides, both in vivo and in votro studies suggested that K145 stimulated insulin dependent Akt phosphorylation and subsequently activates FoxO1 phosphorylation therefore inhibited gluconeogenetic genes expression including PEPCK and G6pase. Our study figures out a potential extent increase the value of developing K145 as therapeutic candidate for diabetes.

Lactobacillus acidophilus alleviates the inflammatory response to enterotoxigenic Escherichia coli K88 via inhibition of the NF-κB and p38 mitogen-activated protein kinase signaling pathways in piglets.

BACKGROUND: A newly isolated L. acidophilus strain has been reported to have potential anti-inflammatory activities against lipopolysaccharide (LPS) challenge in piglet, while the details of the related inflammatory responses are limited. Here we aimed to analysis the ability of L. acidophilus to regulate inflammatory responses and to elucidate the mechanisms involved in its anti-inflammatory activity. RESULTS: The ETEC (enterotoxigenic Escherichia coli) K88-induced up-regulations of IL-1ß, IL-8 and TNF-α were obviously inhibited by L. acidophilus while IL-10 was significantly increased. Moreover, L. acidophilus down-regulated pattern recognition receptors TLR (Toll-like receptor) 2 and TLR4 expression in both spleen and mesenteric lymph nodes of ETEC-challenged piglets, in accompanied with the reduced phosphorylation levels of nuclear factor kappa B (NF-κB) p65 and mitogen-activated protein kinase (MAPK) p38 as well in spleen of ETEC-infected piglets. Furthermore, L.acidophilus significantly increased the expression of the negative regulators of TLRs signaling, including Tollip, IRAK-M, A20 and Bcl-3 in spleen of ETEC-challenged piglets. CONCLUSIONS: Our findings suggested that L. acidophilus regulated inflammatory response to ETEC via impairing both NF-κB and MAPK signaling pathways in piglets.

TLR2/MyD88/NF-κB signalling pathway regulates IL-8 production in porcine alveolar macrophages infected with porcine circovirus 2.

Porcine circovirus 2 (PCV2) is the primary cause of post-weaning multisystemic wasting syndrome, in which it stimulates a strong IL-8 response that is associated with chronic inflammation as well as lesions in the lymphoid organs. However, the mechanism underlying PCV2-induced IL-8 production is still unclear. In the present study, we demonstrated that increased IL-8 expression during PCV2 infection depends on Toll-like receptor (TLR2), but not TLR4 or TLR9 signalling pathways in porcine alveolar macrophages. Moreover, we found that impairment of the MyD88/NF-κB signalling pathway by MyD88 knockdown or NF-κB inhibitors markedly decreased PCV2-induced IL-8 secretion. These results suggest that PCV2 induces IL-8 secretion via the TLR2/MyD88/NF-κB signalling pathway. Therefore, it is important to elucidate the molecular mechanisms of the PCV2-induced inflammatory response.

Effects of Lactobacillus acidophilus dietary supplementation on the performance, intestinal barrier function, rectal microflora and serum immune function in weaned piglets challenged with Escherichia coli lipopolysaccharide.

This study was conducted with a lipopolysaccharide (LPS)-challenged piglet model to determine the effects of diets containing Lactobacillus acidophilus on the performance, intestinal barrier function, rectal microflora and serum immune function. A total of 150 piglets (initial body weight (BW) 7.53 ± 0.21 kg) were allotted to one of the following diets, including a basal diet, a basal diet supplemented with 250 mg/kg Flavomycin, or basal diet plus 0.05, 0.1 or 0.2 % L. acidophilus. On day 28 of the trial, the pigs were given an intraperitoneal injection of LPS (200 µg/kg body weight) followed by blood collection 3 h later. Diets with either antibiotics, 0.1 or 0.2 % Lactobacillus increased (P < 0.05) the final BW and decreased (P < 0.05) feed gain ratio (F/G) compared with the control group. Pigs fed diets containing antibiotic or Lactobacillus had greater average daily gain (ADG) (P < 0.05) than pigs fed the control diet. The rectal content Lactobacillus counts for pigs fed diet containing Lactobacillus were significant higher (P < 0.01) than those fed antibiotic or control diet. Feeding the Lactobacillus diets decreased the Escherichia coli counts of rectal content (P < 0.01). Pigs fed diets containing 0.1 or 0.2 % Lactobacillus decreased serum DAO activity (P < 0.05) compared with pigs fed the control diet. Serum IL-10 concentration was enhanced in pigs fed the diet with Lactobacillus compared to pigs fed the control diet and antibiotic diet. Feeding a diet with Lactobacillus reduced (P < 0.05) IFN-γ concentration compared to the control diet. Inclusion of Lactobacillus in diets fed to pigs reduced TNF-α concentration compared with pigs fed no Lactobacillus (P < 0.05). These results indicate that feeding with L. acidophilus improved growth performance and protected against LPS-induced inflammatory status.

[Clinical evaluation on the preparation of cured root canals with Reciproc and Pathfile rotary instruments].

PURPOSE: To evaluate the effect of cured molar root canal preparation using Reciproc and Pathfile rotary instruments. METHODS: From December 2012 to January 2013, 88 teeth with 273 root canals were randomly divided into three groups. In group A, root canals were prepared with Reciproc and Pathfile. In group B, root canals were prepared with ProTaper and Pathfile. In group C, root canals were prepared with rotary ProTaper. All root canals were filled with continuous wave condensation. The operating time for each root canal, the incidence of instrument separation and postoperative pain were recorded. The efficiency of reparation was analyzed with radiograph. The data was analyzed with SPSS17.0 software package. RESULTS: In group A, the mean operation time was significantly shorter without instrument separation, transportation and ledge. The technique could keep the curvature and flow of curved canals. Postoperative pain was significantly lower, and appropriate filling of root canals was significantly higher than in other groups (P<0.01). CONCLUSIONS: Curved root canal preparation with Reciproc and Pathfile is less time consuming and more convenient with fewer side effects. It is an efficient instrumentation for curved canals and can be used widely.

Expression of Bacillus subtilis MA139 beta-mannanase in Pichia pastoris and the enzyme characterization.

The 1014 nucleotides long gene-encoding beta-mannanase from Bacillus subtilis strain MA139 was cloned using PCR. To obtain high expression levels in Pichia pastoris, the beta-mannanase gene was optimized according to the codon usage bias of P. pastoris and fused downstream of GAP promoter. The reconstituted plasmid pGAP-mann was transformed into P. pastoris X-33 strain to constitutively express beta-mannanase. When cultured at 28 degrees Celsius for 3 days protein yields up to 2.7 mg/mL was obtained with the enzyme activity of up to 230 U/mL. In comparison, wild-type gene product yielded 1.9 mg/mL and 170 U/mL, respectively indicating that the protein yield and enzyme activity were significantly improved by codon modification. After purification, the enzyme properties were characterized. The optimal activity was at pH 6.0 and 50 degrees Celsius. In the pH range of 3.0 to 9.0, beta-mannanase showed above 60% of its peak activity. Among the numerous ions tested copper significantly inhibited the enzyme activity. These results suggested that codon-optimized beta-mannanase expressed in P. pastoris could potentially be used as an additive in the feed for monogastric animals.

Cloning of a beta-1,3-1,4-glucanase gene from Bacillus subtilis MA139 and its functional expression in Escherichia coli.

A gene encoding beta-1,3-1,4-glucanase was cloned by polymerase chain reaction (PCR) from Bacillus subtilis MA139. Sequencing result showed 97% homology to the corresponding gene from Bacillus licheniformis. The open reading frame (ORF) of the gene contained 690 bp coding for a 226 amino-acid matured protein with the estimated molecular weight of 24.44 kDa. The beta-1,3-1,4-glucanase gene was subcloned into an expression vector of pET28a and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a nickel-nitrilotriacetic acid (Ni-NTA) column. The purified beta-1,3-1,4-glucanase demonstrated 24.05 and 12.52 U ml(-1) activities for the substrates of barley beta-glucan and lichenan, respectively, and the specific activities were 728.79 and 379.1 U mg(-1) for them, respectively. The optimal temperature and pH of the purified enzyme were 40 degrees C and 6.4, respectively. When barley beta-glucan was used as the substrate, K (m) was 5.34 mg ml(-1), and K (cat) showed 7,206.71 S(-1), thus the ratio of K (cat) and K (m) was 1,349.67 ml s(-1) mg(-1). The activity of beta-1,3-1,4-glucanase was affected by a range of metal ions or ethylenediaminetetraacetic acid (EDTA).

De novo synthesis, constitutive expression of Aspergillus sulphureus beta-xylanase gene in Pichia pastoris and partial enzymic characterization.

The endo-beta-1, 4-xylanase gene xynA from Aspergillus sulphureus, encoded a lack-of-signal peptide protein of 184 amino acids, was de novo synthesized by splicing overlap extension polymerase chain reaction according to Pichia pastoris protein's codon bias. The synthetic DNA, composed of 572 nucleotides, was ligated into the downstream sequence of an alpha-mating factor in a constitutive expression vector pGAPzalphaA and electrotransformed into the P. pastoris X-33 strain. The transformed yeast screened by Zeocin was able to constitutively secrete the xylanase in yeast-peptone-dextrose liquid medium. The heterogenous DNA was stabilized in the strain by 20-times passage culture. The recombinant enzyme was expressed with a yield of 120 units/mL under the flask culture at 28 degrees C for 3 days. The enzyme showed optimal activity at 50 degrees C and pH 2.4-3.4. Residual activity of the raw recombinant xylanase was not less than 70% when fermentation broth was directly heated at 80 degrees C for 30 min. However, the dialyzed xylanase supernatant completely lost the catalytic activity after being heated at 60 degrees C for 30 min. The recombinant xylanase showed no obvious activity alteration by being pretreated with Na(2)HPO(4)-citric acid buffer of pH 2.4 for 2 h. The xylanase also showed resistance to certain metal ions (Na(+), Mg(2+), Ca(2+), K(+), Ba(2+), Zn(2+), Fe(2+), and Mn(2+)) and EDTA. These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed industry as an additive.